Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reduced folate carrier (RFC) is the dominant influx transporter for antifolates. A major mechanism of antifolate resistance is loss of RFC (SLC19A1) gene expression due to decreased GC-box-dependent transcription. However, despite the poor GC-box binding in multiple antifolate-resistant cell lines, normal Sp1 levels were retained. Here we explored the post-translational modifications that may disrupt Sp1 function. Phospho-affinity purification of nuclear proteins revealed that resistant cells contained approximately 8-fold more phosphorylated Sp1 than parental cells; treatment of nuclear proteins from these cells with alkaline phosphatase restored GC-box binding. As protein kinase A phosphorylates Sp1, resistant cells were treated with various cAMP-reactive agents, revealing no apparent effect on GC-box binding except for the general phosphodiesterase inhibitor IBMX. As cGMP levels also may be affected by IBMX, resistant cells were treated with 8-pCPT-cGMP, resulting in the complete restoration of GC-box binding, luciferase reporter activity, and RFC mRNA levels. This restoration was abolished in the presence of the protein phosphatase 2A inhibitor (PP2A) okadaic acid. Importantly, whereas resistant cells showed multiple phosphorylated Sp1 forms barely detectable in parental cells, treatment with 8-pCPT-cGMP resulted in their elimination; this disappearance, however, was prevented by the copresence of okadaic acid. These findings provide the first evidence that loss of RFC gene expression in antifolate-resistant cells is associated with an inhibitory Sp1 phosphorylation that can be eliminated by a cGMP-dependent activation of PP2A.
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PMID:Loss of Sp1 function via inhibitory phosphorylation in antifolate-resistant human leukemia cells with down-regulation of the reduced folate carrier. 1616 3

Towards utilizing gene-targeted, repopulating mesenchymal stem cells (MSC) to increase osteogenesis, we evaluated the expression of bone-specific promoters during MSC differentiation. Multi-lineage potential of cultured MSC was confirmed by osteogenic, adipogenic and chondrogenic differentiation under controlled conditions. Recombinant adeno-associated virus (rAAV) encoding luciferase under the human cytomegalovirus (CMV), mouse alkaline phosphatase (ALP), Runx-2/cbfa1 (RUNX), osteopontin (OPN), collagen type 1a (COL), and osteocalcin (OCN) promoters was used to transduce mouse MSC. Replicate cultures were maintained undifferentiated or differentiated to osteoblast lineage. Luciferase expression was determined on days 1, 2, 3, 7, 14, or 21 as a measure of promoter activity. Expression of osteogenic markers and mineralization was determined as correlates of osteopoiesis. Results indicated expression from CMV promoter in undifferentiated and differentiated cultures at early stage. However, expression from COL and RUNX promoters was abundant only in differentiating cultures as early as 24 h but declined gradually. Expression from OPN and ALP promoters was evident 24 h following osteogenic differentiation and peaked gradually until 2 weeks before declining. Expression from OC promoter was evident only after 7 days of differentiation but remained until final analysis on day 21. That rAAV transduction of MSC does not induce differentiation was also confirmed by quantitative reverse-transcription polymerase chain reaction (QRT-PCR). The observed stage-specific expression of analyzed promoters was not significant when the MSC were differentiated to adipocytes. Thus, the use of RUNX2 or COL promoter to stably express osteoinductive factors in MSC may allow both self-renewal of modified MSC and enrichment of osteoblast commitment.
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PMID:Determination of osteoprogenitor-specific promoter activity in mouse mesenchymal stem cells by recombinant adeno-associated virus transduction. 1622 39

Reporter genes such as firefly luciferase are common tools to monitor gene expression in various systems. As reporter gene represents the expression level of the gene of interest with its enzyme activity, firefly luciferase is most frequently used because its luminescent activity is highly sensitive and less time consumable for assay. However, since firefly luciferase is expressed internally in the cell, lysis of the cell is a critical step, and thus it is difficult to monitor the gene expression level continuously. In this report, we utilized secretive Vargula hilgendorfii luciferase modified to cell surface displayed one by fusing with human EGFR transmembrane sequence. This modified Vargula luciferase was expressed on cell surface without losing its bioluminescent activity. Co-transfection with secretive alkaline phosphatase showed that the behaviors of cell surface displayed Vargula luciferase and secretive alkaline phosphatase are comparable to each other. Furthermore, the luminescence of a single cell expressing cell surface displayed Vargula luciferase can be monitored by using photon counting CCD camera, which indicates that this reporter gene can monitor gene expression in a single cell without cell lysis.
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PMID:Single cell reporter assay using cell surface displayed Vargula luciferase. 1623 49

Bone morphogenetic proteins (BMPs) control cell fate by regulating gene expression, especially inhibitor of differentiation (Id) genes. This property has been exploited to create a highly sensitive assay for quantification of active BMP. Embryonic mouse cells (C3H10T1/2) were stably transfected with an expression construct (BRE-Luc) containing a BMP-responsive element fused to the firefly luciferase reporter gene. BRE results from a multimerization of distinct sequences elements from a mouse Id1 promoter [15]. The addition of BMP-2 (0.5-100ng/ml) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. This new assay was 100-fold more sensitive than the classical alkaline phosphatase (ALP) activity assay (0.5-1 vs. 50-100ng/ml, respectively) as well as much more rapid (24h vs. 3-6 days, respectively, of BMP treatment). This new assay is specific to BMPs (BMP-2, BMP-4, and BMP7) as evidenced by its relative insensitivity to TGFbeta1, bFGF, and VEGF. Because of its BMP specificity, this rapid, sensitive, nonradioactive, and easily performed assay could be used in monitoring the biological activity of BMP and, eventually, as a cell-based screening assay to identify and evaluate molecules that modulate BMP signaling in cells.
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PMID:An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct. 1630 14

We synthesized four derivatives of 17beta-estradiol (E2) with an azide substitution on a 17alpha-side chain of varying length, namely 17alpha-(azidopropargyl)-3,17beta-estradiol (5), its 17beta-azido derivative (diazide 7), 17alpha-(5-azido-pent-1-ynyl)-3,17beta-estradiol (6) and 17alpha-(azidopentyn-2-yl)-3,17beta-estradiol (10). While most of the derivatives had low (7) or marginal (6 and 10) relative binding affinity (RBA) for both types of estrogen receptor (ERalpha and ERbeta), the RBAalpha and RBAbeta of 5 were practically identical to those of E2. The estrogenic activity of the derivatives was assessed using estrogen-responsive breast (MCF-7) and endometrial cancer (Ishikawa) cells. While 5 was a potent and effective inducer of alkaline phosphatase in Ishikawa cells and 7 was less potent but as effective as 5, 6 was marginally active and 10 was totally inactive in this respect. In the presence of 0.1 nM E2, however, 6 exhibited some ER antagonist activity at the highest concentration tested (1 microM). Similar results were obtained as regards the potency and efficacy of stimulation of MCF-7 cell proliferation and induction of luciferase gene expression in MCF-7:D5L cells, a clone stably transfected with an estrogen-responsive form of the gene. These data suggest that, while 5, 6, 7 and 10 interact with either type of ER in isolation, only 5 and 7 exhibit substantial ER agonist activity in the different estrogen-target cells examined, which could provide for photoaffinity labelling of the receptor in the cell as well as in isolation.
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PMID:High affinity 17alpha-substituted estradiol derivatives: synthesis and evaluation of estrogen receptor agonist activity. 1636 Jul 21

In most developing countries, 70-80% of the population still resort to traditional medicine for their primary health care. This medicine utilises medicinal plants which are traditionally taken as concoction and infusion. The root and stem bark of Millettia griffoniana (Leguminosae), has been reported to contain isoflavonoids, alkaloids, and diterpenoids. The possible benefit of some bioactive isoflavones derived from M. griffoniana prompted us to screen them for estrogenic activity. Six isoflavones and coumarin derived from M. griffoniana (bail) namely, compound nos. 1-6 (Fig. 1) were tested for their potential estrogenic activities in three different estrogen receptor alpha (ERalpha)-dependent assays. In a yeast-based ERalpha assay, all test substances and 17beta-estradiol as endogenous agonist, showed a significant induction of beta-galactosidase activity. The test compounds at the concentration of 5 x 10(-6) M could achieve 59-121% of the beta-galactosidase induction obtained with 10(-8) M 17beta-estradiol (100%). In the reporter gene assay based on stably transfected MCF-7 cells (MVLN cells), the estrogen responsive induction of luciferase was also stimulated by the M. griffoniana isoflavones. In Ishikawa cells, all substances exhibited estrogenic activity revealed by the induction of alkaline phosphatase (AlkP) activity. The estrogenic activities of isoflavones from M. griffoniana could be completely suppressed by the pure estrogen antagonist, ICI 182,780, suggesting that the compounds exert their activities through ERalpha. Although all substances showed estrogenic effects, 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone (7-O-DHF), Griffonianone C (GRIF-C), and 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO) were found to be the most potent of tested substances. In summary, estrogenic activities of the isoflavones derived from M. griffoniana were described for the first time using reporter gene assays and the estrogen-inducible AlkP Ishikawa model.
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PMID:Estrogenic properties of isoflavones derived from Millettia griffoniana. 1642 19

Nutrient and oxygen availability are key metabolic parameters for biopharmaceutical manufacturing. In order to enable mammalian cells to manifest their intracellular nutrient and oxygen levels we engineered a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a robust reporter gene expression readout. Capitalizing on the Streptomyces coelicolor redox control system, consisting of REX modulating ROP-containing promoters in an NADH-dependent manner, we designed a mammalian dual sensor transcription control system by fusing REX to the generic VP16 transactivation domain of Herpes simplex, which reconstitutes an artificial transactivator (REDOX) able to bind and activate chimeric promoters assembled by placing a ROP operator module 5' of a minimal eukaryotic promoter (P(ROP)). When nutrient levels were low and resulted in depleted NADH pools REDOX-dependent P(ROP)-driven expression of secreted (human-secreted alkaline phosphatase; SEAP) or intracellular (Renilla reniformis luciferase; rLUC) reporter genes was high as a consequence of increased REDOX-P(ROP) affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced REDOX-P(ROP) interaction mediated low-level transgene expression in Chinese hamster ovary (CHO-K1) cells. Other molecules (for example, 2,4-dinitrophenol, cyanide or hydrogen peroxide) which are known to imbalance the intracellular NADH/NAD+ poise could also be detected using the REDOX-P(ROP) sensor circuitry. REDOX's sensor capacity (nutrient and oxygen levels) operated seamlessly in transgenic CHO-K1 cell derivatives adapted for growth in serum-free suspension cultures and enabled precise monitoring of the population's metabolic state. As the first genetic metabolic sensor designed for mammalian cells, REDOX may foster advances in process development and biopharmaceutical manufacturing.
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PMID:A genetic redox sensor for mammalian cells. 1647 37

Dimethandrolone (DMA), the 17beta-undecanoic acid ester of dimethandrolone (DMAU; 7alpha,11beta-dimethyl-19-nortestosterone) is a potent androgen currently in development for therapeutic uses in men. Cleavage of the 17beta-ester bond liberates the biologically active DMA. In this study we investigated the activity of DMAU and DMA both in vivo and in vitro. DMAU was active orally in castrate rat bioassays, and when administered sc, a single dose produced prolonged androgenic activity and suppression of LH with sustained circulating levels of DMA. DMA, other 19-norandrogens, and C-19 androgens bound to recombinant rat androgen receptor with high affinity and were equipotent in stimulating luciferase activity (EC50, 10(-10) -10(-9) M) in CV-1 cells cotransfected with a human androgen receptor expression vector and a luciferase reporter plasmid with three hormone response elements. Because various 19-norandrogens are also known to bind to progestin receptors (PR) and to possess progestational activity in vivo, we evaluated the binding affinity of DMA for rabbit PR and recombinant human PR-A and PR-B and its ability to induce PR-mediated transcription and endogenous alkaline phosphatase activity in T47DCO human breast cancer cells. DMA and related 19-norandrogens bound with high affinity to both rabbit and human PR, whereas the less active 11alpha-methyl stereoisomer of DMA and C-19 androgens showed low or negligible binding to PR. In T47DCO cells, 10(-8) M DMA and other 19-norandrogens stimulated transcription of a progestin/glucocorticoid/androgen response element-thymidine kinase-luciferase reporter plasmid to the same extent as R5020, the potent progestin promegestone (EC50, approximately 10(-9) M), but C-19 androgens had no effect. Antiprogestins were potent inhibitors of transactivation and alkaline phosphatase activity induced by DMA and other 19-norandrogens in T47DCO cells, whereas antiandrogens were weak inhibitors. DMA and DMAU also exhibited dose-dependent progestational activity in the estrogen-primed immature female rabbit, as assessed by induction of endometrial gland arborization. The dual androgenic and progestational activities of DMA make it a potential candidate for a single-agent male contraceptive as well as for androgen therapy in men, pending a successful outcome of pharmacokinetic and toxicity studies currently in progress.
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PMID:Dimethandrolone undecanoate: a new potent orally active androgen with progestational activity. 1649 1

The benzothiophene selective estrogen receptor modulators (SERMs), raloxifene and arzoxifene, in the clinic or clinical trials for treatment of breast cancer and postmenopausal symptoms, are highly susceptible to oxidative metabolism and formation of electrophilic metabolites. 4'F-DMA, fluoro-substituted desmethyl arzoxifene (DMA), showed attenuated oxidation to quinoids in incubation with rat hepatocytes as well as in rat and human liver microsomes. Incubations of 4'F-DMA with hepatocytes yielded only one glucuronide conjugate and no GSH conjugates, whereas DMA underwent greater metabolism giving two glucuronide conjugates, one sulfate conjugate, and two GSH conjugates. Phase I and phase II metabolism were further evaluated in human small intestine microsomes and in human intestinal Caco-2 cells. In comparison to DMA, 4'F-DMA formed significantly less glucuronide and sulfate conjugates. The formation of quinoids was further explored in hepatocytes in which DMA was observed to give concentration- and time-dependent depletion of GSH accompanied by damage to DNA, which showed inverse dependence on GSH; in contrast, GSH depletion and DNA damage were almost completely abrogated in incubations with 4'F-DMA. 4'F-DMA shows ligand binding affinity to estrogen receptor (ER)alpha and ERbeta with similarity to both raloxifene and to DMA. ER-mediated biological activity was measured with the ERE-luciferase reporter system in transfected MCF-7 cells and Ishikawa cells, and in MCF-7 cells, proliferation was measured. In all systems, 4'F-DMA exhibited anitestrogenic activity of comparable potency to raloxifene but did not manifest estrogenic properties, mirroring previous results on inhibition of estradiol-mediated induction of alkaline phosphatase activity in Ishikawa cells. These results suggest that 4'F-DMA might be an improved benzothiophene SERM with similar antiestrogenic activity to raloxifene but improved metabolic stability and attenuated toxicity, showing that simple chemical modification can abrogate oxidative bioactivation to potentially toxic metabolites without loss of activity.
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PMID:Chemical modification modulates estrogenic activity, oxidative reactivity, and metabolic stability in 4'F-DMA, a new benzothiophene selective estrogen receptor modulator. 1678 Mar 56

Activity of secreted alkaline phosphatase (SEAP) produced by transfected cells is rapidly down-regulated by endoplasmic reticulum (ER) stress independent of transcriptional regulation. This phenomenon was observed in a wide range of cell types triggered by various ER stress inducers. The magnitude of the decrease in SEAP was proportional to the extent of ER stress and inversely correlated with the induction of endogenous ER stress markers grp78 and grp94. In contrast to SEAP, activity of secreted luciferase was less susceptible to ER stress. The decrease in SEAP activity by ER stress was caused by abnormal post-translational modification, accelerated degradation and reduced secretion of SEAP protein. In transgenic mice constitutively producing SEAP, systemic induction of ER stress led to reduction in serum SEAP. In these mice, administration with lipopolysaccharide caused rapid, transient decrease in serum SEAP activity, and it was correlated with up-regulation of grp78 in several organs including the spleen, lung, kidney, liver and heart. These results elucidated for the first time a possible involvement of transient, systemic ER stress in endotoxemia and provided evidence for usefulness of ER stress responsive alkaline phosphatase for real-time monitoring of ER stress in vitro and in vivo.
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PMID:Real-time detection and continuous monitoring of ER stress in vitro and in vivo by ES-TRAP: evidence for systemic, transient ER stress during endotoxemia. 1687 67


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